Biomolecular applications of single-molecule measurements: kinetics and dynamics of a single-enzyme reaction

Matt Paige; David P. Fromm; William E. Moerner

doi:10.1117/12.463828

28 March 2002 Biomolecular applications of single-molecule measurements: kinetics and dynamics of a single-enzyme reaction

Matt Paige, David P. Fromm, William E. Moerner

Author Affiliations +

Proceedings Volume 4634, Methods for Ultrasensitive Detection II; (2002) https://doi.org/10.1117/12.463828
Event: High-Power Lasers and Applications, 2002, San Jose, California, United States

Abstract

In this work we describe preliminary experiments in which we have used ultra-sensitive fluorescence microscopy to observe the dynamics of individual enzyme molecules acting upon a substrate. The enzyme, (beta) -galactosidase from E.coli, is specifically immobilized onto a glass substrate while maintaining its functionality. The immobilized protein degrades a fluorogenic substrate to produce a fluorescent product, whose generation can be observed in real time. Individual copies of (beta) -galactosidase can be observed for many minutes, allowing the measurement of a large number of successive substrate turnover events. A rudimentary analysis of these turnovers using autocorrelation functions is presented, and a strong heterogeneity in reaction rates between different molecules is observed. In addition, the challenges inherent in successful surface immobilization of proteins for single-molecule experiments are discussed.

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Matt Paige, David P. Fromm, and William E. Moerner "Biomolecular applications of single-molecule measurements: kinetics and dynamics of a single-enzyme reaction", Proc. SPIE 4634, Methods for Ultrasensitive Detection II, (28 March 2002); https://doi.org/10.1117/12.463828

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